A Bifunctional Fluorogenic Rhodamine Probe for Proximity-Induced Bioorthogonal Chemistry

Article abstract of DOI:10.1002/chem.201703607

Bioorthogonal reactions have emerged as a versatile tool in life sciences. The inverse electron demand Diels–Alder reaction (DA_inv) stands out due to the availability of reactants with very fast kinetics. However, highly reactive dienophiles suffer the disadvantage of being less stable and prone to side reactions. Herein, we evaluate the extent of acceleration of rather unreactive but highly stable dienophiles by DNA-templated proximity. To this end, we developed a modular synthetic route for a novel bifunctional fluorogenic tetrazine rhodamine probe that we used to determine the reaction kinetics of various dienophiles in a fluorescence assay. Under proximity-driven conditions the reaction was found to be several orders of magnitude faster, and we observed almost no background reaction when proximity was not induced. This fundamental study identifies a minimally sized fluorogenic tetrazine dienophile reactant pair that has potential to be generally used for the visualization of biomolecular interactions with temporal and spatial resolution in living systems.

Full text of DOI:10.1002/chem.201703607

A Journal of
Accepted Article
Title: A Bifunctional Fluorogenic Rhodamine Probe for Proximity-
Induced Bioorthogonal Chemistry
Authors: Philipp Werther, Jasper S. Möhler, and Richard Wombacher
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To be cited as: Chem. Eur. J. 10.1002/chem.201703607
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Chemistry - A European Journal
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A Bifunctional Fluorogenic Rhodamine Probe for
Proximity-Induced Bioorthogonal Chemistry
╪
╪
M.Sc. Philipp Werther , M.Sc. Jasper S. Möhler and Dr. Richard Wombacher*
Institut für Pharmazie und Molekulare Biotechnologie, Ruprecht-Karls-Universität
Heidelberg, Im Neuenheimer Feld 364, 69120 Heidelberg, Germany.
E-mail: wombacher@uni-heidelberg.de
╪
These authors contributed equally
*Corresponding author
1
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Abstract
Bioorthogonal reactions have emerged as a versatile tool in life sciences. The inverse electron
demand Diels-Alder reaction (DAinv) stands out due to the availability of reactants with very
fast kinetics. However, highly reactive dienophiles suffer the disadvantage of being less stable
and prone to side reactions. Here, we evaluate the extent of acceleration of rather unreactive
but highly stable dienophiles by DNA-templated proximity. To this end, we developed a
modular synthetic route for a novel bifunctional fluorogenic tetrazine rhodamine probe that
we used to determine the reaction kinetics of various dienophiles in a fluorescence assay.
Under proximity-driven conditions the reaction was found to be several orders of magnitude
faster, and we observed almost no background reaction when proximity was not induced. This
fundamental study identifies a minimally sized fluorogenic tetrazine dienophile reactant pair
that has potential to be generally used for the visualization of biomolecular interactions with
temporal and spatial resolution in living systems.
Graphical Abstract
2
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Introduction
One of the most important criteria in the synthesis of complex organic molecules is
[1]
[2]
“
chemoselectivity”, a term first coined by Barry M. Trost in 1973 . IUPAC defines
chemoselectivity as the “preferential reaction of a chemical reagent with one of two or more
different functional groups”. In the more recent past, chemists have started to perform
chemical reactions in biological environments for labeling purposes, to modify or to control
the function of biomolecules. Once more, this challenges researchers to solve even more
sophisticated problems of chemoselectivity: In the overcrowded and dense cellular milieu, a
multitude of endogenous molecules carrying a vast number of chemical functionalities, small
nucleophilic molecules, (metal) ions, reactive oxygen species etc. are present and potentially
interfere with introduced artificial chemistry. Reactions that exhibit satisfying
chemoselectivity in biosystems have been termed “bioorthogonal” by Carolyn R. Bertozzi in
[3]
2
003. To truly accomplish a bioorthogonal reaction, additional requirements have to be
fulfilled. Starting material, product, by-products should not be harmful to the biological
system and must not interfere with cellular or tissue processes. Moreover, the formed inert
chemical bond must not perturb native biomolecule function in an unwanted way. Another
crucial aspect is the kinetic profile of the reaction: The reaction must be reasonably fast in the
aqueous milieu (physiological pH, high salt content, reactive nucleophiles) of biological
[4]
systems. Several reactions that match these criteria have been (re-)invented by chemical
[5]
biologists. These include the Staudinger ligation and the catalyst-free strain-promoted azide
alkyne cycloaddition (SPAAC)[6] as well as the inverse electron demand Diels-Alder
cycloaddition between 1,2,4,5-tetrazines and (strained) olefins (DAinv).[7]
Based on superior characteristics, DAinv has emerged as bioorthogonal chemistry of choice in
[8]
many applications. Contrary to normal electron demand Diels-Alder reactions, DAinv employs
weak Michael acceptors as dienophiles which are rather stable against endogenous
nucleophiles. Moreover, the release of dinitrogen as the only by-product drives the
equilibrium. The outstanding advantage of the DAinv is its extremely fast kinetics (k
2
up to
1
06 M-1s-1 [9]) As a fortunate circumstance, aqueous reaction media increase rates compared
to other protic (and aprotic) solvents.[10] Rapid reactions are crucial if fast cellular processes
are under investigation and to diminish competing slower side reactions. The fastest known
rates in bioorthogonal chemistry have been achieved with ring-strained olefins in DAinv
.
However, despite of its popularity in recent applications for biomolecular modification,
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limitations have emerged on using this chemistry. High reactivity in chemistry comes with the
cost of selectivity, as reactive species are less stable and therefore open to alternate reaction
pathways. This has come to display in DAinv as well. Broadly used trans-cyclooctenes (TCOs)
are prone to isomerize to less reactive cis-cyclooctenes.[11] Other dienophiles undergo
elimination post-cycloaddition[12] and (electron-deficient) tetrazines can react with
endogenous nucleophiles.[13] If DAinv is applied in protein labeling, this leads to a low labeling
density, which is a drawback for biophysical applications like localization microscopy. Although
numerous interesting approaches aim to stabilize TCOs by structural modification,[9, 14] it is
highly challenging to maintain high reactivity at the same time. Taken altogether, the key
feature of DAinv, the rapid reaction rates, potentially puts the bioorthogonality requirement
“chemoselectivity” at risk. One way to overcome this stalemate situation is the use of stable
and less reactive DAinv-reactants in combination with a secondary, proximity-inducing process
that accelerates reaction rates.
In the concept of proximity-induced rate enhancement, molecular recognition creates an
intramolecular situation and therefore increases the active concentrations of the reactants
and hence the overall rate of the reaction. Proximity-accelerated reactions are not only of
prime interest in the design of improved bioorthogonal reactions, but have been used
extensively for sensing and imaging of nucleic acids.[15] Staudinger ligation[16] (e.g. QSTAR
probes), copper catalyzed azide alkyne cycloaddition (CuAAC),[17] native chemical ligation,[18]
nucleophilic substitution (S
)[19] as well as photoligation[20] are notable chemistries templated
N
by nucleic acids. Two recent publications that utilize templated turn-over reactions elucidate
the advancement this field has come to already: Kool et al. used Staudinger chemistry to
detect RNA at concentrations of 10 pM and single point deletions while the Winssinger group
reached enzyme-like rates in the PNA-templated photocatalytic reduction of azides.[21]
Beyond that, ligand-directed reactions are of special interest for the development of covalent
drugs[22] and in protein labeling schemes . Oligonucleotide-mediated DAinv have been
developed by the Devaraj group for the reaction turn-over sensing of mRNA.[24] They
employed a tetrazine fluorogenic BODIPY dye and vinyl-ether-caged cyanine fluorophore,
both inducing strong fluorescence after undergoing DAinv and enabling a reaction turnover
[23]
(Figure 1).
4
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Figure 1: Schematic overview of templated chemistry using dienophiles and tetrazine fluorophore
conjugates or dienophile-caged fluorophores respectively. *References marked with asterisks
report initial template-mediated cycloadditions, followed by elimination resulting in reaction
turnover.
In the light of the overall relevance of proximity-induced chemistry in life sciences and in
particular of the significance and current limitations of DAinv for bioorthogonal chemistry, it is
of special interest to evaluate the scope of DAinv in proximity-induced chemistry. To this end,
we present the design and synthesis of a highly fluorogenic tetrazine rhodamine probe that is
equipped with an additional functionality for further conjugation to biomolecules. The novel
dye was ideally suited to study the rate-acceleration of rather unreactive and stable
dienophiles in nucleic acid-templated DAinv with a direct and sensitive fluorescence read-out.
This work represents a comprehensive evaluation of dienophile-reactivity under non-
mediated and template-mediated conditions and gives valuable information and indications
for future developments of highly bioorthogonal reactions. Moreover, the presented
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fluorogenic tetrazine rhodamine dye equipped with a biomolecule targeting site is
predestined for advanced studies of biological processes.
Results and Discussion
Modular Synthesis of a Bifunctional Tetrazine Rhodamine
To investigate proximity-directed DAinv with a fluorogenic tetrazine reporter, we envisioned a
synthetic strategy based on recent work from our laboratory. In this approach, tetrazinyl
benzaldehydes were reacted with diarylethers (or silanes) in Friedel-Crafts reactions to
assemble fluorogenic tetrazine (Si)rhodamines which we showed to be suitable for no-wash
protein labeling inside living cells.[25] The design of the fluorophore probe followed the
principles: (1) minimal interchromophore distance between the fluorophore and the tetrazine
for strong fluorescence quenching and for small probe size, (2) well-suited fluorophore classes
for biological application (3) modular synthetic access under mild conditions. Due to the
modular nature of our strategy, a biarylether can alternatively be used in the Friedel-Crafts
reaction as nucleophile that carries an additional carboxylic function for bioconjugation. The
convergent synthesis route (Scheme 1) opens the door to easily accessible fluorogenic
tetrazine rhodamine dyes that can be conjugated to a biomolecule with an intact tetrazine
moiety still efficiently quenching the fluorophore. Subsequently, the tetrazine can be used for
a second conjugation by DAinv resulting in a strong increase of fluorescence, equivalent to a
small molecule-based “light-up” cross linker. In this work, a novel bifunctional reporter
(Rhod-Tz-NHS) was used to investigate a set of different dienophiles of various reactivity in
proximity-accelerated DAinv. For the synthesis of Rhod-Tz-NHS, biarylether 5 was obtained via
I
[26]
a Cu -mediated coupling reaction of aryliodide 2 with phenol 4 in 60% yield (Scheme 1, I).
Precursors 2 and 4 were obtained from the commercially available starting materials 1 and 3
by either halogen exchange reaction with subsequent reduction (for 2) or simultaneous
reduction and ethylation (for 4).[26a, 27] Diethylene glycol was converted to 7 over two steps[28]
resulting in a PEG-2 chain linker that enhances hydrophilicity of the final fluorogenic probe. To
be compatible with Lewis acid-mediated Friedel-Crafts reaction, the carboxylic acid was tert-
butyl ester-protected (Scheme 1, II). Tetrazine benzylic alcohol was afforded from
hydroxymethyl benzonitrile (39%) and subsequently oxidized to benzaldehyde 9 using Dess-
Martin periodinane (85%) (Scheme 1, III).[25]
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Scheme 1: Three buildings blocks (I-III) enable the modular synthesis (IV) of the bifunctional dye
Rhod-Tz-NHS.
After attaching 7 to the biarylether 5 (84%), the resulting modified biarylether was converted
with benzaldehyde 9 to tetrazinyl rhodamine S5 via AlBr -mediated Friedel-Crafts reaction and
3
subsequent oxidation with p-chloranil (36%). Deprotection of S5 yielded Rhod-Tz in 77% yield.
Rhod-Tz-NHS was obtained by using a standard protocol (EDC·HCl, N-hydroxysuccinimide) and
subsequent HPLC purification yielded the target fluorogenic probe in the purity needed for
further experiments (66%) (Scheme 1, IV). We would like to highlight particularly that the
modular nature of the herein developed route (building blocks I-III in Scheme 1) allows the
independent modification of each building block providing the synthetic flexibility needed for
further tailor-made probes. To the best of our knowledge, Rhod-Tz-NHS represents the first
fluorogenic tetrazine crosslinker based on a rhodamine fluorophore. First, the probe can be
conjugated to an amine of a biomolecule, presenting the tetrazine for a second fluorogenic
bioorthogonal DAinv
.
Photophysical Characterization of the Bifunctional Probe
Overall, the herein described synthesis represents a novel access to a highly water-soluble
fluorogenic probe which carries two orthogonal reactive groups for bioconjugation. Most
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importantly, the tetrazine fluorophore probe exhibits strong fluorescence enhancements
upon conversion in DAinv (Figure 2a). Before applying the novel tetrazine probe in the
investigation of proximity-accelerated DAinv, we first evaluated its photophysical and
fluorogenic properties (Figure 2b-d). Absorption and emission spectra of Rhod-Tz were
recorded in aqueous phosphate buffered saline (PBS) and the absorption maximum was
4
-1
-1
determined to λabs = 559 nm (ε = 4.4·10 M cm ), the emission maximum to λem = 582 nm
Figure 2b). The fluorescence quantum yield Φ = 0.017 is in accordance with previously
reported tetrazine quenched rhodamines.[25]
(
f
Figure 2: Photophysical properties and fluorogenicity of Rhod-Tz. (a) Reaction with BCN.
Cuvettes with Rhod-Tz were irradiated with a handheld UV-lamp (365 nm) w (left) and w/o (right)
BCN. (b) Normalized absorption and emission spectra. (c) Emission spectra w/o BCN and after
addition of 10 eq BCN. (d) Time course measurement. (a: 10 µM, b-c: 1 µM, each in PBS).
Fluorogenicity of Rhod-Tz was quantified to 34-fold fluorescence enhancement when
converted with (1R,8S,9s)-bicyclo[6.1.0]non-4-yn-9-ylmethanol (BCN) as model dienophile.
Ten equivalents of BCN led to completion of fluorescent product formation after 40 minutes
(Figure 2d). This “turn-on factor” is slightly improved, compared to recently published
fluorogenic tetrazine rhodamines with ultra-short interchromophore distance (1.6 fold
higher).[25] Furthermore, the water-soluble dye is highly stable in aqueous buffer at room
temperature and no degradation was observed over 12 hours. The fluorescence quenching
efficiency is in general dependent on the interchromophore distance and a number of short-
distance tetrazine fluorophore conjugates have been reported.[25, 29] The exact quenching
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mechanism for these fluorogenic tetrazine probes is still under debate, as no thorough
physicochemical investigation has been reported yet.
Dienophiles for Proximity-Induced DAinv
Various dienophile classes have found widespread application in bioorthogonal reactions with
tetrazines in DAinv. These range from unstrained and slowly reacting primary olefins[30] to ring-
strained and more reactive dienophiles such as norbornenes[7b] and cyclopropenes.[31] Highly
strained cyclic alkynes (e.g. cyclooctynes, e.g. BCN) and olefins (prominently trans-
cyclooctenes, e.g. TCO, sTCO) are among the dienophiles reaching the fastest reaction
kinetics. Due to the extremely rapid reaction rates, especially TCO-derivatives have emerged
6
-1 -1
[9]
to dienophiles of choice in DAinv (k ≤ 10 M s in aqueous media ). However, their
2
applicability is constrained to a certain degree as they degrade under prolonged storage and
thiol exposure leads to metabolic instability under serum conditions.[9, 11a] In this context,
tremendous efforts have been made to develop stable and still highly-reactive TCO-
dienophiles.[9, 14] Moreover, the rather large size of TCOs may perturb native biomolecule
function and the hydrophobicity of TCO-derivatives has been linked to increased fluorescent
background in imaging and the necessity of elaborate washing procedures.[14f, 32] Bypassing
TCO-derivatives entirely, an obvious approach would be to use less reactive, smaller and more
stabilized dienophiles. Their intrinsic slow reaction rates in DAinv can be overcome by inducing
spatial proximity between the reactants. A promising model system for proximity-
acceleration, as has been shown by the Devaraj group for DAinv,[24a] is the molecular
recognition of nucleic acid hybrids. DNA-templated chemistry was also chosen in this study to
evaluate the extent of proximity-acceleration on dienophiles of varying reactivity.
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Figure 3: Dienophiles with a broad spectrum of reactivity in DAinv and high stability in biological
environments.
To this end, we selected a set of stable dienophiles ranging from low to medium reactivity
which react rapidly only under proximity-driven conditions (Figure 3). These include
-2
-1 -1 [30a, 30b]
unstrained linear α-olefins (k
2
~10 M s ,
11, 12), cyclopentene (13, 14) and
~1.9 M s-1 [7b], 15), all of which exhibit reaction rates at least six
-
1
norbornene derivatives (k
2
orders of magnitude lower than TCO-derivatives. In particular, the accelerated reaction of
slow α-olefin tags is of interest due to their minimal size and high stability.[30c] Beyond the
varying reactivity of the chosen dienophiles (Figure 3), our selection enables to study the
effects of conformational freedom which supposedly has a profound influence on DAinv
-
reactivity under proximity-mediated conditions. On this account, different linker lengths were
chosen at the examples of primary alkenes (11, 12) and cyclopentene derivatives (13, 14). The
commercially available unsaturated organic acids (11 to 15) were converted to their
NHS-esters, (see SI) which can be used to attain dienophile-modified nucleic acids needed for
template-driven DAinv
.
Conjugation of the Fluorogenic Diene and Dienophiles to DNA
The concept of proximity-driven DAinv obviously carries the potential to put the reaction under
control of a biomolecular interaction. Therefore, the dienophiles of choice should only react
with the tetrazine (fluorogenic Rhod-Tz) when brought in close proximity. With the herein
reported conjugatable fluorogenic tetrazine the biomolecular interaction can be translated
into a fluorescent read-out signal. DNA hybridization is the most well-defined and previously
extensively applied biomolecular interaction to create molecular proximity. Briefly, we studied
the effect of proximity on DAinv by using three components: (1) a 14mer oligonucleotide 3’-
modified with the fluorogenic Rhod-Tz, (2) 14mer 5’-modified dienophiles and (3) various
template sequences to examine the effects of sequence variation and length (Figure 4a-c). To
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yield components (1) and (2), Rhod-Tz-NHS and the dienophiles (11 to 15-NHS, SI) were
attached to the 3’ or 5’-amino-modified oligonucleotides by means of amide coupling
chemistry. We used flexible linker structures (Figure S1) between the amine functionalities
and the 14mers to ensure the necessary conformational flexibility for the templated DAinv. A
ten to thirty-fold excess of the NHS-activated dienophiles or Rhod-Tz-NHS respectively was
used to reach complete conversion of the amino-modified 14mers within two to four hours.
The reaction mixtures were monitored by HPLC, subsequently purified on a preparative scale
and the identity of the afforded 14mer-dienophiles and 14mer-Rhod-Tz was confirmed via
LCMS (Table S1, Figure S1).
Figure 4: Components for proximity-accelerated DAinv. Structure of 14mer-Rhod-Tz (a) and 14mer-dienophiles
(
3
b). (c) Varying template sequences to study the influence of base gaps between the DAinv-reactants (28mer to
1mer-g3), of spatial separation through hybridization (29mer-r) or of a mismatch three bases apart from DAinv
reaction site (29mer-m).
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Figure 5: (a) Schematic representation of the assay: 14mer-Rhod-Tz, 14mer-dienophiles, template sequence and
resulting cycloaddition product. (b) Reversed template places reactive groups on opposite ends of the formed hybrid
(29mer-r).
Complementary template sequences were designed to hybridize with 14mer-dienophile and
1
4mer-Rhod-Tz. The exact template sequences are shown in Figure 4c. Templates 28mer,
9mer-g1, 30mer-g2 and 31mer-g3 were used to study the effect of varying distances
2
between DAinv-reactants. Moreover, a template with a reversed sequence (29mer-r) serves as
a control where hybridization places the reactive groups on opposite ends of the double
strand and thus prevents successful product formation. A randomly inserted mismatch in
29mer-g1 provided the template 29mer-m which can be used to study the effect of imperfect
hybridization on the proximity-enhanced reaction.
Rate Enhancement of DAinv by Template-Induced Proximity
To study the influence of template-induced proximity on the reaction rates of different
dienophiles in DAinv, the above described tetrazine and dienophile-modified oligonucleotides
were mixed in absence and presence of an oligonucleotide template strand at equimolar
concentrations (1 µM) in aqueous PBS. The fluorogenic tetrazine allows to easily follow the
reaction progress by recording the changes in fluorescence over time. To that purpose, the
template DNA-strand was added to the fluorogenic probe-modified oligonucleotide
14mer-Rhod-Tz and the measurements were started upon addition of the dienophile-
modified oligonucleotides 14mer-dienophiles (Figure 5a). Fluorescence intensity was
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recorded every 45-135 s (depending on the speed of the reaction) over a total of four hours
excitation wavelength λex = 559 nm, emission wavelength λem = 582 nm). To exclude that
photo bleaching affects the measurements, we analyzed a control sample containing solely
4mer-Rhod-Tz in PBS with the same fluorometer settings. No significant changes in intensity
(
1
could be observed over the entire experiment. The diagrams in Figure 6a-e show the reaction
kinetics of the different 14mer-dienophiles with 14mer-Rhod-Tz in DAinv in the presence of
different templates. The template sequences were designed to be complementary to both
14mer sequences with either no or up to three additional bases in between (see Figures 4c
and 5a). We supposed that, upon hybridization, the resulting gaps (g1, g2, g3) in the final
double strand would position the dienophile and tetrazine at various distances and
orientations, thereby creating different spatial proximity.
Whereas we did not observe any reaction in the absence of template over the entire period
of four hours for 14mer-11 to 14, we did observe a slight increase in fluorescence when using
the 14mer-15 (Figure S2). From the change in fluorescence the consumption of 14mer-15 in
DAinv can be calculated to below 0.6%. This indicates that even norbornene, the most reactive
dienophile of this study, exhibits only negligible background under non-templated conditions
at the concentrations used. Obviously, the reaction only proceeded in the presence of a
complementary template strand. When using the template 29mer-r (see Figure 5b), which
contains the complementary 14mer-sequences in a reversed manner, we did not observe any
reaction, either (Figure S3). This is an expected result as, with this template, hybridization
positions the dienophile and tetrazine distant from each other, not able to meet for reaction.
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Figure 6: Kinetics of template-mediated DAinv between 14mer-dienophiles and 14mer-Rhod-Tz.
a-e) Using various template sequences for the 14mer-dienophiles. (f) Comparison of all
4mer-dienophiles in proximity-induced DAinv using template 29mer-g1. Both reactants and
(
1
templates were used in equimolar quantities in PBS (1 µM).
The template-mediated DAinv with the templates 28mer, 29mer-g1, 30mer-g2, 31mer-g3 in all
cases showed strong rate enhancement over the non-templated reaction (Figure 6a-e). While
the fastest kinetics can be observed with the g1-template, the g2- and g3-template showed
the expected slower kinetics, due to the increasing distance between dienophile and tetrazine.
This trend is consistent for all dienophiles used in the study and is in accordance with other
DNA-templated proximity systems reported in literature,[24a] where a single nucleotide gap
has also been shown to be favorable. The 28mer-template, which theoretically leads to the
closest positioning of both reactants, clearly showed slower reaction kinetics than the
29mer-g1-template. This observation might result from the type of modification we chose for
the attachment of the reactants to the 5’-and 3’-position of the respective 14mers (see Figure
S1). The modification of the 14mer at the 5’-phosphate with the dienophile respectively at the
3’-hydroxy with the tetrazine can lead to sterically disfavored reactant orientation when used
with the 28mer template. This indicates the importance of spatial orientation of reactants for
the probability of collision, a prerequisite for reaction. Interestingly, we also see differences
in the total change of fluorescence over time for the different templates while using the same
14mer-dienophile and 14mer-Rhod-Tz. Although this effect is not dramatic, the differences
are clearly visible from the fluorescence after completion of the reaction. It should be noted
that the formed Diels-Alder product is identical in each experiment, though hybridized to
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different template strands. The hybridization of the ligated 14mer-strands to the different
templates might lead to differences in the degrees of freedom for the formed fluorescent
cycloadduct, resulting in slightly different fluorescence properties. Figure 6f summarizes the
reaction kinetics of all five 14mer-dienophiles in the presence of the 29mer-g1 template. The
dienophiles that are more reactive in DAinv also show faster kinetics in the proximity–induced
reaction. Again we observed different fluorescence intensities after full conversion. However,
the differences of the fluorescence intensities were significantly higher compared to the
previous results when only the template sequence was variable. It has to be noted that the
formed Diels-Alder products in Figure 6f are different, which is likely to result in fluorophores
with different quantum yields. This has previously been described for fluorogenic tetrazine
conjugated probes.[29c] Moreover, DAinv between tetrazines and olefins results in a mixture of
tautomeric and diastereomeric dihydropyridazines as well as pyridazines.[7a, 7b, 33] We found
the strongest fluorescence increase after full conversion to be 42-fold for the dienophile
14mer-14. Full conversion of the ligation process (indicated by reaching a plateau) was verified
-
and analyzed by ESI TOF HRMS and no side products could be identified. Furthermore, we
analyzed the aqueous stability (PBS, pH 7.4, 30°C) of the oligonucleotide fluorogenic tetrazine
rhodamine conjugate by HR-MS and HPLC, which was found to be stable with no significant
degradation of the tetrazine moiety for at least 12 hours.
As expected, the fastest reaction was observed with 14mer-15 reaching the maximum
fluorescence after six minutes. 14mer-14 showed the second highest reactivity and the
respective DAinv-product possessed the highest observed turn-on value (Figure 6f).
Additionally, we analyzed the influence on the reaction kinetics when using a template with a
single mismatch in the sequence (29mer-m) (Figure 4c). The mismatch, three bases apart from
the ligation site, caused a deceleration of the DAinv compared to the gap-template 29mer-g1.
Apparently, the introduced mismatch led to a slightly delayed reaction, likely due to a
perturbed hybridization process. However, the effect of the mismatch on the reaction rate
was less pronounced compared to templates 30mer-g2 or 31mer-g3.
An overview of the above-noted maximal fluorescence turn-on values of templated DAinv is
shown in Figure 7a. The fold increases were calculated by dividing the final fluorescence
intensity by the respective baseline intensities. The lowest turn-on values were obtained for
the primary alkenes 14mer-11 and 14mer-12 (7 to 19-fold). 14mer-13 and 14mer-15 exhibited
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turn-on values between 24 and 35-fold. The highest fluorescence increase was determined to
2-fold for the 29mer-g1-templated reaction of 14mer-14.
4
Figure 7: (a) Fluorescence fold increase after reaction completion in proximity-induced DAinv (up to 43-fold).
b) Overview of calculated apparent first-order rate constants kapp (spanning almost two orders of magnitude).
(
To determine the apparent first-order rate constants (kapp) from the reaction kinetics, an
exponential function was applied to the measured fluorescence data. The obtained data is
summarized in Figure 7b and Table S3. The lowest kapp were determined for primary olefin
-4 -1
1
4mer-11 (kapp [10 s ] = [0.7±0.2] to [2.5±0.3]) and cyclopentene 14mer-13
-4 -1
(
kapp [10 s ] = [0.8±0.1] to [2.5±0.2]). Interestingly, the rates of primary olefin 14mer-12
-4 -1
(k
app [10 s ] = [2.2±0.3] to [3.6±0.7]) were higher than those of cyclopentene 14mer-13 and
-4 -1
were of the same order of magnitude as cyclopentene 14mer-14 (kapp [10 s ] = [1.6±0.2] to
4.6±0.3]). Two major factors have to be taken into account here: First, the electron densities
[
of the alkenes differ depending on the positon of the amide group. Secondly, length and
conformational flexibility of the reactants influence the probability for a collision that leads to
a chemical reaction. The longer carbon chain in nonenoic acid of 14mer-12 might have played
a role for the increased reaction rate compared to 14mer-11 and 14mer-13 and the increased
dienophile flexibility surpassed the higher reactivity in DAinv of cyclopentene derivatives. The
higher rate of the cyclopentene moiety in 14mer-14 compared to the one in 14mer-13 could
16
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Chemistry - A European Journal
10.1002/chem.201703607
arise from the higher dienophile-mobility and lower electron deficiency (α-dialkyl-substituted
alkene) in 14mer-14. Expectedly, kapp significantly increased for norbornene 14mer-15, the
most reactive dienophile used in this study, to the highest herein determined values
-4 -1
(k
app [10 s ] = [22±1] to [46±3]). The determined kapp are in accordance with reported first-
order rate constants for DNA-templated reactions.[21b, 24, 34] The half-lives ranged from
6±6 min (14mer-11) to 2.5±0.9 min (14mer-15) in DAinv with 29mer-g1 as template (Table
4
S4). Taken together, the kinetic analysis illustrates the considerable extent to which reaction
rates differ from non-templated DAinv-conditions (see above, negligible to 0.6% conversion) to
templated conditions. Under proximity-induced acceleration, the dienophile-reactivity as well
as mobility and length of linker structures greatly influence the reaction rate. Moreover, the
choice of the template (fully complementary, gap length at DAinv-site) results in varying
reaction rates. The template-length and linker structure-dependency of the reaction kinetics
are in accordance with previous reports.[15a, 24a] Still, the reactivity of the dienophile was the
determining factor for the reaction rates observed in the template-mediated DAinv
.
In summary, the kinetics of dienophiles were investigated in terms of their performance in
DAinv using a templated model system. Stable dienophiles with poor kinetic characteristics did
not react at micromolar concentrations without a DNA template strand present. However, the
hybridization-induced reactions between the tetrazine probe and dienophiles were
-3
-4 -1
significantly accelerated and kapp ranging from 10 to 10 s were determined. Remarkably,
the templated fluorogenic bioorthogonal reaction led to an increase in fluorescence intensity
of up to 42±1-fold. This observed fluorescence turn-on was higher compared to the reaction
between Rhod-Tz and BCN (34±2-fold). The reason for this could be environmental effects of
the oligonucleotides in addition to different quantum yields of the formed cycloadducts.
Furthermore, we suggest the use of cyclopentene derivative 14 in future fluorogenic
proximity-enhanced biorthogonal experiments. It equates to a minimal sized tag and, as our
study shows, it combines reasonable kinetics with a high fluorescence turn-on (42-fold).
Conclusion
In this work we report the effect of proximity on DAinv-biorthogonal chemistry by evaluating
dienophiles of varying reactivity in a well-defined DNA-templated assay. We developed a
highly convergent, modular synthesis route towards easily modifiable multi-functional
fluorogenic xanthene-based dyes ready for bioconjugation. The synthetic route provides
17
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Chemistry - A European Journal
10.1002/chem.201703607
access to future tailor-made probes through simple modification of the used building blocks.
The synthesized probe Rhod-Tz-NHS represents the first fluorogenic tetrazine crosslinker
based on a rhodamine fluorophore. For the purpose of this study, the novel bifunctional
tetrazine-dye served us as fluorescence turn-on probe, providing us the direct read-out
necessary to study different dienophiles in proximity-induced bioorthogonal DAinv. Our study
shows that the lack in reactivity in DAinv of highly stable dienophiles with poor kinetic
characteristics can be overcome by DNA-templated chemistry induced proximity.
Fluorescence signal generation resulted solely from proximity-induced reaction, while
background reactivity was absent at micromolar concentrations. The concept of proximity can
be highly beneficial to overcome stability problems of commonly used reactants in
bioorthogonal chemistry while maintaining the rapid reaction rates needed for cellular
applications. Furthermore, the results indicate the potential of our fluorogenic tetrazine dye
Rhod-Tz for the visualization of biomolecular interactions with temporal and spatial resolution
in vitro as well as in living systems.
Acknowledgements
R.W. acknowledges funding from the Deutsche Forschungsgemeinschaft DFG (SPP1623, WO
1888/1-2). We thank Dr. Achim Wieczorek for scientific advice, Mathis Baalmann and Michael
Ziegler for helpful comments to the manuscript. We gratefully acknowledge Prof. Dr. Andres
Jäschke for constant support.
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